Purification and characterization of Pz-peptidase B, a neutral metalloendopeptidase from bovine spermatozoa.
We have demonstrated previously that the Pz-peptide synthetic substrate is cleaved by two distinct spermatozoal peptidases, Pz-peptidases A and B. To facilitate further investigations, Pz-peptidase B was purified from bovine spermatozoa. The soluble extract from 81 grams of washed epididymal spermatozoa was fractionated by a five-step procedure consisting of anion-exchange, lectin affinity, hydrophobic interaction, chromatofocusing, and gel filtration chromatography. This method yielded 1 mg of 170-fold purified Pz-peptidase B with a 26% recovery. The purified Pz-peptidase B was electrophoretically homogeneous and possessed a monomeric molecular weight of 90,700. Isoelectric focusing revealed microheterogeneity with pIs ranging from 5.02 to 5.09. Pz-peptidase B was irreversibly inactivated at pH 3.5 or below, and activity was reduced at moderate ionic strengths. Hydrolysis of the Pz-peptide was maximal at pH 7.5. Pz-peptidase B was strongly inhibited by a metal chelator and phosphoramidon. Reactivation of metal-depleted enzyme by various metal ions suggested that Pz-peptidase B was a zinc metallopeptidase. Pz-peptidase B hydrolyzed a wide variety of synthetic substrates and physiologically activity peptides at the amino side of hydrophobic amino acids. These results established that Pz-peptidase B should be classified as a neutral metalloendopeptidase. Overall, the properties of Pz-peptidase B were very similar to those of previously described neutral metalloendopeptidases implicated in degradation of regulatory peptides.
Lessley
B A
BA
Oklahoma State University, Department of Physiological Sciences, Stillwater 74078.
Garner
D L
DL
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002417:Cattle; D003300:Copper / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D006020:Glycopeptides / Q000494:pharmacology; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007526:Isoelectric Point; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008666:Metalloendopeptidases / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight; D009532:Nickel / Q000494:pharmacology; D013094:Spermatozoa / Q000737:chemistry*; D015032:Zinc / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D006020:Glycopeptides; D003300:Copper; D009532:Nickel; D010450:Endopeptidases; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; D008274:Magnesium; D015032:Zinc; C008890:phosphoramidon